The goal of this proposal is to investigate the mechanisms by which protein ubiquitination regulates cellular resistance to oxidative stress. Increased exposure to oxidants combined to the accumulation of toxic protein aggregates is the cause of several neurodegenerative diseases such as Parkinson's and Alzheimer's. Oxidative stress is a very prominent type of stress induced by exposure to diverse environmental factors such as ionizing radiation, heat, and pollutants. Oxidative stress damages several biomolecules including lipids, DNA and proteins, potentially resulting in cell death. To prevent cell death and disease progression, cells use ubiquitination as the signal for removing damaged proteins through the proteasome and thus preventing aggregation. However, many non-degradative roles for ubiquitination have been described which are signaled by different lysine (K) ubiquitin chains when conjugated to target proteins. Using yeast S. cerevisiae as a model, we found that the unconventional K63 polyubiquitin chain accumulates strongly and dynamically, and impacts cellular viability in response to oxidative stress. Using innovative proteomics approaches, we identified many ribosome proteins as targets of K63 ubiquitination. However, the mechanisms by which K63 ubiquitination regulates cellular viability are still unknown. Therefore, in Aim1 system-wide approaches will be employed to investigate how K63 ubiquitination affects protein expression (K99). In Aim 2, interaction analysis will be performed to define the mechanisms that regulate the specificity of the K63 ubiquitin pathway in response to oxidative stress (K99/R00). Further, it has been demonstrated that mammalian cells accumulate K63 ubiquitin in response to oxidative stress, but the evolutionary conservation of this redox pathway is completely unknown. In Aim 3, the redox K63 ubiquitin system will be taken to mammalian cells to investigate overall conservation by mapping this pathway to the stress response in neuronal cells (R00). The outcome of this project will unravel novel mechanisms to increase cell tolerance to stress depending on selective ubiquitination. My entire career focused on studying different regulatory aspects of the ubiquitin proteasome system in response to stress. During the K99 mentored phase of this award, I will obtain training in genomics and computational techniques that will enable the investigation of one more layer of cellular signaling mediated by ubiquitin. NYU offers an ideal combination of intellectual environment and multidisciplinary facilities for th development of the K99 phase of this project. My long-term career goal is to establish my independent research group that will combine systems-wide methodologies with molecular biology and genetic approaches to decode the ubiquitination signals in response to stress. This award offers a unique opportunity to achieve this goal by enabling precise training and career guidance from my mentors and committee for my transition to independence. Understanding the multiple ubiquitin roles in cellular response to stress and the specificity of each distinct pathwa will provide groundbreaking insights into innovative strategies for treatment and early diagnosis of many damage-related disorders.